PROJECT SUMMARY/ABSTRACT Huntington's disease (HD) is a devastating and fatal neurodegenerative disorder caused by the expansion of a polymorphic CAG repeat in the HTT gene that triggers cell death with a specificity towards neurons in the striatum and cortex. Although the underlying genetic mutation was discovered 25 years ago, there is still no cure or effective treatment. Notably the expanded repeat undergoes further expansion over time in somatic cells, particularly in the brain regions affected in the disorder, implying that somatic HTT CAG length increases in target tissues contribute to HD pathogenesis. This is strongly supported by genome-wide association studies in which mismatch repair genes, known to drive somatic CAG expansion in HD mouse models, have emerged as significant disease modifiers in patients. Overall, a wealth of data support somatic CAG length in target tissues/cells as a critical determinant of the rate of disease onset, and that slowing the rate of somatic expansion would have therapeutic benefit. Thus, understanding the factors that contribute to somatic CAG instability is of critical importance as these may provide novel targets for therapeutic intervention. Both trans- acting and cis-acting factors can contribute to the instability of trinucleotide repeat tracts. In this study, with the goal of uncovering novel modifiers of CAG instability, we will use innovative state-of-the-art CRISPR-based approaches to identify candidate modifiers of HTT CAG instability that may act by influencing local chromatin/DNA structure at the HTT locus. In Aim 1, using a candidate gene approach targeting genes that control chromatin/DNA structure, we will use adeno-associated viruses (AAVs) to deliver guide RNAs to tissues of HD knock-in mice expressing Cas9, and will determine the impact of inactivation of these genes on HTT CAG instability. In Aim 2, we will take an unbiased, locus-specific proteomics approach towards identifying factors that are associated with the expanded HTT CAG repeat locus. Using patient-derived and control induced pluripotent stem cells (iPSC) we will use CRISPR-Cas9 targeting coupled with local protein biotinylation to enrich the HTT locus and identify associated proteins using mass spectrometry. Together, these complementary approaches, using both accurate genetic mouse models and patient cells, provide tools to generate novel candidate genes/proteins that may alter CAG repeat length by acting in cis to the repeat, which will be pursued in subsequent R01-phase mechanistic studies. In the longer term, these studies will impact directly on the development of therapeutics targeting the CAG repeat mutation itself, with real potential for disease modification.